The aim of this project is to further elucidate the mechanisms of action of intracellular messengers such as calcium ion, cAMP, cGMP and protein kinases on electrical properties of nerve cell membranes, in particular the membrane channels. Experiments will be done on molluscan ganglion cells and cultured vertebrate excitable cells, utilizing voltage clamp in conjunction with injection or perfusion methods, and single-channel recording with a patch-clamp pipette. In parallel with these approaches, a major effort will go into the application of a new means of rapidly and uniformly perturbing the intracellular concentration of Ca2+. This will make use of high-affinity Ca-complexing ligand with Ca-buffering properties similar to EGTA, but that undergoes increased dissociation when irradiated with strong light of appropriate wavelength. Such a photo-modulated Ca buffer will allow changes in intracellular pCa rapidly and uniformly with light pulses. We will also utilize caged nucleotides which are converted from an inactive to an active form by flash photolysis, liberating the agent rapidly in the intracellular milieu or in a cell-free system such as the isolated membrane on the end of a patch-clamp pipette. These methods for modulating Ca and nucleotide agonist concentrations with illumination are intended to open the way for approaches such as high-resolution relaxation analysis that are not available to us with present methods of manipulating these intracellular messengers. In addition, we will continue conventional approaches to the role of second messengers in the inactivation of Ca channels, the activation of K channels, and the generation of pacemaker activity.